This document provides an overview of tissue fixation techniques. It defines fixation as a process that preserves tissues in a state close to how they appeared when living. This is achieved by preventing autolysis and maintaining cellular morphology. The document discusses various types of fixatives including aldehydes, alcohols, and oxidizing agents. It also covers the aims of fixation, how different fixatives work, commonly used fixatives for different tissue and cellular components, and potential artifacts. Fixation is essential for histological examination and aims to maintain tissues for further analysis.

1. 1

2. Dr NAVEEN
KUMAR
I MDS,OMFP

3.  Glossary of terms
 Introduction
 Definition
 Types of fixation
 Classification of fixatives
 Effects and aim
 Reaction of fixatives
 Commonly used fixatives
 Factors affecting fixation
 Fixation for specialized techniques
 Fixation artefacts
 summary
 References
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4. Autolysis – lysis or dissolving of cells by enzymatic
action, probably as a result of rupture of lysosome. The
group of enzymes – cathepsins
Putrefaction – breakdown of tissue by bacterial action,
often with the formation of gas.
Precipitation – a process in which a solid is separated
from a suspension or solution.
Denaturation - a major change from the original native
state without alteration of the molecule’s primary
structure.
Osmolality - the molality of an ideal solution of a non
dissociating substance that exerts the same osmotic
pressure as the solution being considered.
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5. Additive: they chemically link or bind to the
tissue and change it.
Non- additive: they act on the tissue without
chemically combining with the tissue. Eg:
alcohols
Coagulant : it will allow solutions to penetrate
into the interior of the tissue very easily
Non-coagulant: they act by creating a gel like
barrier that makes the solution more difficult
to penetrate to the interior of the tissue.
Birefringence - the double refraction of light in
a transparent, molecularly ordered material
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6.  It is difficult to conduct histochemical investigations upon
living
 To study the microanatomy
 For good histological preparations
 Tissues should be fixed early
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7. Fixative (Dorland’s):
“ A fluid, often a mixture of several reactive chemicals , into which
histological or cytological specimens are placed so that, by processes such as
denaturation and cross-linking of proteins, autolysis is prevented, the
specimen is hardened to withstand further processing and the specimen is
preserved in a close facsimile of the living state in regard to both cellular
morphology and the location of sub cellular constituents.” 7
Fixation:
“ A process by which the constituents of the cells or tissues are
fixed in a physical and chemical state so that they will withstand
subsequent treatment with various reagents with a minimum loss,
distortion or decomposition.”

8.  Three types of fixation
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Heat fixation
Perfusion
Immersion

9. Micro anatomical
• 10% formol saline
• 10% neutral buffered
formalin
• Zenkers solution
• Bouin’s solution
• Rossman’s fluid
• Formol calcium
Cytological
• 1. Nuclear fixatives
• glacial acetic a-
affinity for nuclear
chromatin.
• pH≤ 4.6
• Flemming’s
• Carnoy’s
• Newcomer’s
• Clarke’s
• 2. Cytoplasmic
fixatives
• glacial acetic a -
destroys mitochondria
and Golgi.
• pH ≥ 4.6.
• Kelly flemmings
• Regauds’s fluid
• Orth’s fluid
Histochemical
• Formol saline 10%
• Absolute ethyl alcohol
• Acetone
Based on mode of action COMPOUND FIXATIVES
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10. MICROANATOMICAL
Formol calcium (Baker, 1944)
Formalin ------------- 10 mg
Calcium chloride ---- 2g
Water -----------------to 100ml
Buffered formalin
Formalin------------------------------- 10ml
Acid sod. Phos. Monohydrate--- 0.4g
Anhydrous disodium phos. ------ 0.65g
Water ---------------------------------to 100ml
Buffered formol sucrose (Holt & Hicks, 1961)
Formalin -------------------------10ml
Sucrose ---------------------------7.5g
M/15 phos. Buffer----------to 100ml
-preserve- fine structure, phospholipids, enzymes
Acetic – alcoholic- formalin
Formalin---------5ml
Glacial acetic A----5ml
70% alcohol---------90ml
-excellent- glycogen
-Fix- nuclear protein
- rapid, 5mm thick- 4hrs
Formol calcium (Lillie,1965)
Formalin...............10ml
Calcium acetate....2g
Water...................to 100ml
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11. Zenker’s fluid
Mer. chlr.-------5g
Pot. Dichromate---
2.5g
Sod. Sulphate---- 1g
Dist. Water----- 100ml
Glacial acetic A -----
5ml
-Rapid & even
penetration
-Fx -12hrs;3mm- 2 to
3 hrs
Zenker’s formol(Helly’s fluid)
Formalin --5ml
-Fx – bone marrow, spleen
- fx – 6-24hrs
Bouin’s fluid
Picric acid sat. aq. Soln-----75ml
Formalin(40% formaldehyde)—25ml
Glacial acetic acid------------------ 5ml
-Penetartes -rapid, even
-Brilliant staining – trichome methods
-Glycogen
-Fx -24hrs
-2-3mm thick – 2-3 hrs
Rossman’s fluid
Formalin-----10ml
Abs. ethyl alc
Sat. picric acid---
90ml
- carbohydrates
MICROANATOMICAL
Heidenhain’s susa
Mercuric chloride....4.5g
Sod. Chloride..........0.5g
Trichloro acetic acid..2g
Acetic acid.............4ml
Formalin ..............20ml
Dist. Water..........to 100ml
-excellent fx- routine biopsy
-brilliant staining; good- cytological detail
-penetration- rapid & even
-tissues left 24hrs- bleaching, hardening
CARBOHYDRATES
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12. NUCLEAR FIXATIVES
Carnoy’s fluid
Abs. alcohol--- 60ml
Chloroform---- 30ml
Glacial acetic A– 10ml
-exclnt nuclear fixation
-preserve – nissil substance, glycogen
-Very rapid
-fx – 1-2 hrs; 2-3mm thick- 15min
Newcomer’s fluid
Isopropanol---------60ml
Propionic acid-------30ml
Petroleum ether-----10ml
Acetone----------------10ml
Dioxane----------------10ml
-fx- chromosomes
-Fx- 12-18hrs; 3mm thick- 2-
3hrs
Flemming’s fluid
1% aq. Chromic acid—15ml
2%aq. Osmium tetroxide–
4ml
Glacial acetic acid ---- 1ml
-Poor & uneven penetration
-2mm thick- 12hrs
Clarke’s fluid
Abs. alcohol------- 75ml
Glacial acetic acid--- 25ml
-rapid, good nuclear fixation
-Excellent- smears 12

13. CYTOPLASMIC FIXATIVES
Champy’s fluid
3% pot. Dichromate ------ 7ml
1% chromic acid------------7ml
2% osmium tetroxide----4ml
-Freshly made
-Penetration –poor, uneven
-Preserves- mito, fat, lipids
-2mm thick- 12hrs
Regaud’s fluid
3% pot. Dichromate-------80ml
Formalin------------------20ml
-freshly prepared
-Penetrates evenly, rapidly
-overharden –tissue
-Mitochondia
-Fx- 24hrs; 3-4mm thick- 4-
6hrs
Muller’s fluid
Pot. Dichromate------ 2.5g
Sod. Sulphate---------1g
Dist. Water-----------to
100ml
-rarely used
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14. Based on chemical agents
1. cross- linking fixatives / aldehydes
-formaldehyde, glutaraldehyde
2. protein denaturing fixatives/ coagulants
- acetic acid, methyl alcohol, ethyl alcohol
3. oxidizing agents
- osmium tetroxide, potassium permanganate, potassium
dichromate
4.Other cross linking agents
- carbodiimides
5.Miscellaneous
- mercuric chloride, picric acid, non-aldehyde –containing
fixatives, dye stuffs
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15.  Autolysis/ putrefaction
 Change in shape/ vol.
 Dessication/ shrinkage of tissue
 Rigidity
 Penetration
 Clear staining
 Tissue- living state
 Semifluid Semisolid
 Optical differentiation
AIM
 To preserve tissue
 To permit
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16. :
 Maintain morphology- stabilize proteins
Cross-links
Gel
Retain cellular const.
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1.Aldeh
ydes
2.
Oxidizin
g agents
3.
Mercuri
c
chloride
4.Micro
wave

17. Change – physical & chemical -DNA & RNA
Eg: mercury , chromium salts
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1. Aldehydes
-Room temp. – x
-↑temp. – uncoiling
DNA(65◦), RNA (45◦)
2. Alcohols
-Commonly used
-Removes histone proteins
-Extract DNA & RNA

18. :
Variable structure, activity – difficult to
analyze
Conventional hp tech.- lipids removed;
cryostats / frozen sections.
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1. Aldehydes
>React –
phospholipids,
unsaturated fatty
acids,
>Double bond is
attacked
2. Mercuric chloride
>React -highly unsaturated
compounds- complexes
>Ultrastructural demo- post
fixation – Imidazole Osmium
tetroxide
>Tannic acid- retain lipids

19.  No single fixative- satisfactory
 Alcoholic – Glycogen
 Ultrastuctural studies- tannic acid, acetyl pyridium
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20. 10% NBF – Common
proteins:
aqueous solution
methylene hydrate (first step)
reacts with proteins side chains
hydroxy methyl side groups (characteristic
reaction)
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21. Advantages Disadvantages
Cheap, easy to prepare, rel.
stable
May cause dermatitis
Allows subsequent appl. Of
most staining tech. without
spl. Preliminary procedures
Fumes might irritate
Frozen sections- easily
prepared
May cause asthma
Staining for fat – easily
carried out
Formation of pigments
Penetrates tissues
reasonably
Does not cause excessive
hardening / brittle
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22. Osmium tetroxide Glutaraldehyde Mercuric chloride
• Hydrophobic &
hydrophilic
• Nucleic acids*
• Clumping of DNA-
prevented by post
fixation KMnO4 / ca++
, tryptophan during
fixation
• Limited penetration to
tissues
• Secondary fixative –
EM
• Stain lipids – frozen
sections
• Tissue swelling –
reversed by
dehydration / adding
NaCl
• Black staining
• Bi functional aldehyde
• Extensive cross linking
for collagen
• Slow penetration of
fixative – any tissue
must be small (0.5mm
max.)
• Increased background
staining
• Usage: 4% glut in
phosphate buffer at
pH7.4
• Secondary fixative
• Reacts – histidine,
thiol, phosphate,
hydroxyl groups
• Carboxyl – X
• Protein precipitant
• Penetration – rapid &
uneven
• Produces H+ ions- soln
more acidic
• Poor ultra structural
preservation
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23. Chromic acid Potassium
dichromate
Picric acid
Dissolving anhydrous
chromic trioxide with
distilled water
Fixes cytoplasm
without
precipitation
Explosive- dry,
Stored under
layer of water
Precipitates
proteins -
picrates
advantage
s
Precipitates- proteins
Preseves- carbohydrates
Hydrolyses DNA
•Preserves
phosphatides-
mitochondria
•Gives brilliant
contrast
•Penetrtion rapid
•preserves
disadvanta
ges
Well washed – running
water
Well washed –
running water
Shrinkage
Corrosion
Cutting
brownish
mercury
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24. 24
ALCOHOL FIXATIVES
Absolute alcohol
Methanol
ethanol

25. Target Fixative of choice Fixative to avoid
Proteins NBF,
paraformaldehyd
e
Osmium tetroxide
Enzymes Frozen sections Chemical
fixatives
Lipids Frozen sec, glut,
osmium tetroxide
Alcoholic fixative,
NBF
Nucleic acids Alcoholic fixatives Aldehydes
Muco
polysaccharides
Frozen sections Chemical
Biogenic amines Bouin’s soln, NBF
Glycogen Alcoholic fixatives Osmium tetroxide25

26. Spray fixatives:
-alcohol based
-aerosol spray cans
-fix cell smears on slides
-water soluble wax- barrier
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Vapour fixatives
-Fix cryostat cut sections
-Formaldehyde- heating paraformaldehyde 50C-
80C
-Acetaldehyde- 80C for 1-4 hrs
-Glutaraldehyde- 80C for 2min-4hrs
POST / SECONDARY FIXATION
-2 fixatives in succession
-Buffered formaldehyde with mercuric chloride
-For EM, after glut, post fix with Osm. tet
Advantages Disadvantages
-sections- cut easily - extra cost
-stain more brilliantly -toxicity
- mitochondria

27.  Buffers and pH
 Penetration (depth 𝐷 = 𝐾√𝑡)
 Duration
 Temperature
 Concentration
 Osmolality
 Volume
 Additives 27

28. Immunohistochemistry
-demonstration of antigen-
fixative and IHC method
-prompt fixation – consistent
results
-Poor fixation – loss of
antigenicity / diffusion
-Eg: 10% NBF, Bouin’s, Formal
mercury
Enzyme histochemistry
-controlled fixation
-Destroys oxidative enzymes
-Hydrolytic enzymes – prefixed in
cold(4◦C) formal calcium
Frozen sections
-Formalin fixed biopsies-
rinsed,
-Immersed in 15-20% sucrose
for 1-8 hrs at 4◦C to replace water
before freezing, improve sectioning
Electron microscopy
-Glutaraldehyde conc.
Between 1.5% and 4%
- osmium tetroxide conc. 1%
or 2%
Flow cyometry
-1% paraformaldehyde- blood cells
-Buffered formaldehyde- fixative of choice
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29. Formalin Mercury Chromic oxide
Colour Brown / blackish brownish black Yellow- brown
Rarely seen
Found blood rich tissues Tissues fixed
with mercury
containing
fixatives
Fixed with chr.,
dichromate
Morphology micro crystalline,
bi refringent
Extracellular
crystal, mono
refringent
Extracellular
and mono
refringent
Removal 10% ammonium
hydroxide in 70%
ethyl alcohol
Lugol’s iodine
and bleaching
with weak
sodium
thiosulfate(hypo)
1% acid alcohol
29Starch – talcum powder - gloves- Maltese cross
configuration – PAS +ve

30.  The possible mechanism of fixation by honey, sugar & jaggery
Fructose present in honey, sugar & jaggery
Low pH
Breakdown to form aldehydes
Aldehydes cross-link with tissue amino acids
(Similar to the action of formaldehyde)
Tissue fixation
30

31. 31

32.  John D Bancroft, Marilyn Gamble – Theory and Practice of Histological
Techniques – 6th edition
 A. Culling – Hand book of histopathological and histochemical techniques
– 3rd edition
 D.J.Cook – Cellular Pathology – 2nd edition
 Steven G. Silverberg - Principles and practice of surgical pathology and
cytopathology -3rd edition
 P. Chakraborty – Practical pathology
 Lynch’s medical laboratory technology – S.S.Raphel,W.B.Saunders
- 4th edition
 Shankargouda Patil, Premalatha B R, Roopa S Rao, Ganavi B S -
Revelation in the Field of Tissue Preservation – A Preliminary Study on
Natural Formalin Substitutes: J Int Oral Health 2013; 5(1):31-38.
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